Download Unearthing the Roles of Imprinted Genes in the Placenta

Placenta 30 (2009) 823–834
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Unearthing the Roles of Imprinted Genes in the Placenta
F.F. Bressan a, T.H.C. De Bem a, F. Perecin a, F.L. Lopes b, C.E. Ambrosio a, F.V. Meirelles a, M.A. Miglino c, *
a
Department of Basic Sciences, Faculty of Animal Sciences and Food Engineering, University of São Paulo, Pirassununga, Brazil
Department of Human Genetics, McGill University, Montreal, Canada
c
Department of Surgery, Faculty of Veterinary Medicine and Animal Sciences, University of São Paulo, São Paulo, Brazil
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Accepted 22 July 2009
Mammalian fetal survival and growth are dependent on a well-established and functional placenta.
Although transient, the placenta is the first organ to be formed during pregnancy and is responsible for
important functions during development, such as the control of metabolism and fetal nutrition, gas and
metabolite exchange, and endocrine control. Epigenetic marks and gene expression patterns in early
development play an essential role in embryo and fetal development. Specifically, the epigenetic
phenomenon known as genomic imprinting, represented by the non-equivalence of the paternal and
maternal genome, may be one of the most important regulatory pathways involved in the development
and function of the placenta in eutherian mammals. A lack of pattern or an imprecise pattern of genomic
imprinting can lead to either embryonic losses or a disruption in fetal and placental development.
Genetically modified animals present a powerful approach for revealing the interplay between gene
expression and placental function in vivo and allow a single gene disruption to be analyzed, particularly
focusing on its role in placenta function. In this paper, we review the recent transgenic strategies that
have been successfully created in order to provide a better understanding of the epigenetic patterns of
the placenta, with a special focus on imprinted genes. We summarize a number of phenotypes derived
from the genetic manipulation of imprinted genes and other epigenetic modulators in an attempt to
demonstrate that gene-targeting studies have contributed considerably to the knowledge of placentation
and conceptus development.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Epigenetics
Genomic imprinting
Knockout
Placentation
Transgenesis
1. Introduction
In mammals, embryo development and survival, as well as
a successful pregnancy, are dependent on the establishment of
a functional maternal–fetal interface. This connection is initiated
during the primary contact of the embryo, followed by embryo
implantation, which is characterized by fetal trophoblast cell
invasion into the maternal endometrium, and it culminates with
the generation of the chorioallantoic placenta [reviewed by [1]].
Together, these processes are referred to as placentation [2].
The phenomenon of genomic imprinting has been demonstrated extensively to play a key role in fetal development and
placentation [3,4]. Although the majority of imprinted genes are
expressed in extraembryonic tissues, there is little information
available on the mechanisms by which such mono-allelic gene
expression regulates placental growth, development and function
[5,6].
Continuous research on placentation and the myriad mechanisms controlling this process is needed to clarify the embryonic–
endometrial interactions, and the use of animal models has
contributed greatly to this study [7]. In particular, genetically
modified animals have provided much of the knowledge on the
genetic control of placental development [8]. In fact, the use of
transgenic models has enabled the creation and analysis of gene
regulation assays; the discovery of new roles for genes in placentation; and, most importantly, it has contributed to our understanding of developmental and perinatal pathologies in animals
and humans. In the present review, we address the epigenetic
events involved in embryogenesis, focusing on imprinted genes
and the knowledge generated by transgenic models as tools to
increase our understanding of the roles that imprinted genes play
in placentation and early development.
2. Epigenetics and development
* Corresponding author. Tel.: þ55 11 30917690.
E-mail address: [email protected] (M.A. Miglino).
0143-4004/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.placenta.2009.07.007
The placenta is the first organ to be formed during pregnancy. It
is responsible for the establishment of vascular connections
between mother and conceptus and allows for the exchange of gas,
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F.F. Bressan et al. / Placenta 30 (2009) 823–834
nutrients and waste. This organ is involved in immune protection of
the fetus and also produces the hormones needed to support fetal
development [9].
The creation of an appropriate maternal environment for fetal
development depends on the proper functioning and development
of the trophoblast cells, which require the well-coordinated
expression of many transcription factors, cell cycle regulators,
growth factors, cytokines and surface receptors [reviewed by
[10,11]]. Embryogenesis and placentation are particularly prone to
perturbations in gene expression because these processes depend
on a complex cascade of events [12,13]. Any disruption to the wellorchestrated expression of these regulatory factors may lead to
placental disorders, causing undesirable phenotypes or even
precocious deaths in animals or humans [9].
Following fertilization, a single-cell zygote forms a multicellular
organism comprised of more than 200 different cell types [14,15].
The development of lineage-specific cells begins with the differentiation of the trophoblast lineage and the inner cell mass [16].
This event depends on epigenetic modifications that control the
expression of particular genes, allowing cells to develop and
differentiate into specific cells and tissues [17].
Epigenetics can be defined as the heritable changes in gene
expression that are not caused by the changes in DNA sequence
[18]. The best studied epigenetic mechanisms are DNA methylation
and histone post-translational modifications, which interact with
each other and also with regulatory proteins and non-coding RNAs
[reviewed by [19]].
The paternal genome is actively demethylated within a few
hours of fertilization, while the maternal genome is demethylated
passively during the first cleavages in a species-dependent manner.
This demethylation, however, spares imprinted genes [20], which
must be maintained throughout development without being
‘‘de novo’’ reprogrammed during the pre-implantation stages [21].
Imprinted genes are expressed selectively from either the
paternal or maternal allele. This specialized form of gene regulation
is necessary for normal development [22,23], as discussed below. In
paternally imprinted genes, the paternal allele is epigenetically
modified, preventing its transcription and leading to mono-allelic
maternal expression [18,24]. The same happens to the maternally
imprinted genes, in which the paternal allele is solely expressed.
These selectively expressed genes are believed to have an important role in the allocation of maternal resources to fetal growth
[25,26].
Imprinted genes are found throughout the mammalian genome,
though their occurrence is not random. These genes tend to be
found in clusters that contain DNA sequences that are rich in CpG
nucleotides. These specific regions, called imprinting control
regions (ICRs), are characterized by epigenetic marks, mainly DNA
methylation and histone modifications, which influence the
binding affinity of transcription activators/suppressors and recruit
chromatin remodeling enzymes to locally change the structure and
function of chromatin [27]. The existence of control regions
suggests that genomic imprinting may be controlled not only at the
single gene level but at the level of the chromosome [28].
Epigenetic marks present in single parental copies of imprinted
regions are responsible for differential gene expression. Interestingly, the maintenance of imprinting has been recently inferred to
depend more on repressive histone methylation than on DNA
methylation in the placenta [6,29].
3. Genomic imprinting and placental development
Approximately 200 genes are imprinted in the mammalian
genome [30]. More than 70 imprinted genes in mice and at least 50 in
humans have already been reported in the current literature (http://
www.mgu.har.mrc.ac.uk/imprinting, http://www.geneimprint.com,
http://igc.otago.ac.nz). In most genes, the imprinting status is
conserved between mouse and human [25] and in some genes the
imprinted status is reported to be conserved also in other species, i.e.,
cattle [31–34]. As summarized in Table 1, imprinted gene expression
can be found in the placenta, the fetus, or both, independently of the
parental origin of the expressed allele, and may be widespread or
specific to certain cell types [4]. Although imprinted gene functions
are generally essential for the proper development and function of
the placenta, as well as for fetal growth [6], some of these genes have
not been reported to be related to development. It is important to
note, however, that imprinted genes can show spatial-temporal
expression [35]. Their expression window during development,
therefore, may be narrow enough to cause the imprinted characteristic to be difficult to recognize.
The placenta is one of the most important sites of imprinted
gene action [[36] reviewed by [37]]. Although placentation displays
species-specific variation [2], the genomic imprinting phenomenon
is conserved amongst eutherian mammals, especially primates,
rodents and ruminants [6,38].
According to the conflict hypothesis [39,40], paternally
expressed genes enhance fetal growth, while maternally expressed
genes suppress fetal growth. One evolutionary explanation for this
hypothesis would be that by restricting fetal growth, females can
have a longer reproductive lifespan, assuring their reproductive
success. In contrast, having more numerous and stronger progeny is
advantageous for males. The conflict hypothesis achieved some
confirmation through observations made with mouse genome
manipulation. Androgenote mice, which contain only paternal
DNA, have poorly developed embryonic components but better
developed extraembryonic tissues, whereas gynogenotes show the
opposite phenotype [41].
It is important to note that both the accurate establishment of
genomic imprints and the correct maintenance of genomic
imprints during embryogenesis are essential for normal embryonic/placental development [42]. Epimutations affecting
imprints can arise during imprint erasure, which occurs when
germ cells migrate to the gonads in pre-natal stages, during
either the imprint establishment that takes place during gametogenesis or imprint maintenance throughout the life of the
organism [43,44].
A clear example of epigenetic disturbance in development is the
interference caused by assisted reproductive techniques (ARTs).
These techniques likely interfere with imprint establishment
(manipulation of gametes) or imprint maintenance (manipulation
of pre-implantation embryos; [43]).
4. Imprinted genes control mammalian development
Insulin-like growth factor 2 (Igf2) was one of the first imprinted
genes to be discovered [45]. Igf2 and its receptor, Igf2r, are essential
during fetal–placental development [46]. While the former is
a maternally imprinted gene that codes for a growth factor involved
in fetal and placental growth in mice and humans, the latter is
a maternally expressed gene in mice involved in Igf2 degradation.
Although recent studies demonstrated that IGF2r is not imprinted
in humans [47,48], the relationship between these genes brings
strength to the conflict theory [49,50].
Igf2, together with H19, which is an imprinted non-coding
transcript, is located in a cluster of imprinted genes in mouse
chromosome 7, syntenic to human chromosome 11p15.5 [51,52]. A
region upstream of H19 regulates imprinted expression of both of
these genes [53]. The establishment and maintenance of DNA
methylation in the Igf2/H19 DMR is acquired during spermatogenesis in the male germ cells; however, the DMR from the female
F.F. Bressan et al. / Placenta 30 (2009) 823–834
825
Table 1
Imprinted gene expression reported in mouse development.
Gene
Aliases
Chromosome
location
Preferentially
imprinted allele
Name
References
Gatm
Nnat
Nesp
AT
Peg 5
Central 2
Distal 2
Distal 2
Paternal
Maternal
Paternal
L-Arginine:glycine amidinotransferase
Neuronatin
Neuroendocrine secretory protein
Distal 2
Maternal
Distal 2
Maternal
Neuro endocrine secretory
protein antisense
Guanine nucleotide binding protein,
alpha stimulating
[140] (Extraembryonic tissues)
[141] (Fetal brain)
[142,143] (Embryonic and
extraembryonic tissues)
[143,144] (embryonic tissues)
Distal 2
Maternal
Distal 2
Distal 2
Maternal
Paternal
Guanine nucleotide binding protein,
alpha stimulating, ‘extra large’
Malignant T-cell amplified sequence 2
Histocompatibility 13
Proximal 2
Maternal
Scm-like with four mbt domains 2
Nespas
Gnas
Gs-alpha
Gnasxl
Mcts2
H13
SPP
Sfmbt2
Calcr
Mit1/Lb9
Clr
Proximal 6
Proximal 6
Paternal
Maternal
Calcitonin receptor
Mest-linked imprinted transcript 1
Sgce
e-SG
Proximal 6
Maternal
Sarcoglycan, epsilon
Peg10
Edr, HB-1, Mar2,
MEF3L, Mart2, MyEF-3
Proximal 6
Maternal
Paternally expressed gene 10
Ppp1r9a
Pon3
Pon2
Asb4
Proximal
Proximal
Proximal
Proximal
Paternal
Paternal
Paternal
Paternal
Mest/Peg1
Proximal 6
Maternal
Neurabin
Paraoxonase 3
Paraoxonase 2
Ankyrin repeat and suppressor
of cytokine signalling
Mesoderm specific transcript
Copg2
Proximal 6
Paternal
Proximal 6
Proximal 6
Maternal
Paternal
Coatomer protein complex subunit
gamma 2
Copg2 antisense
Kruppel-like factor 14
Kvlqt1as
Proximal 6
Proximal 7
Proximal 7
Distal 7
Maternal
Maternal
Paternal
Maternal
Nucleosome assembly protein 1-like 5
Zinc-finger gene 264
Zinc-finger gene 3 from imprinted domain
Kvlqt1 antisense
Pw1, End4, Gcap4, Zfp102
Ocat
Proximal
Proximal
Proximal
Proximal
Paternal
Paternal
Maternal
Maternal
Imprinted zinc-finger gene 3
Imprinted zinc-finger gene 1
Paternally expressed gene 3, probably Pw1
Ubiquitin-specific processing protease 29
Copg2as
Klf4
Nap1l5
Zfp264
Zim3
Kcnq1ot1
Zim2
Zim1
Peg3
Usp29
Ube3a
Epfn, Klf14, epiprofin, BTEB5
Znf264
6
6
6
6
7
7
7
7
Hpve6a, E6-AP ubiquitin
protein ligase
snoRNA MBII-85, Snord116
Peg4, HCERN3
Central 7
Paternal
E6-Ap ubiquitin protein ligase 3A
Central 7
Central 7
Maternal
Maternal
Peg6
ns7, nM15, NDNL1, Mage-l2
Central
Central
Central
Central
7
7
7
7
Maternal
Maternal
Maternal
Maternal
Prader–Willi chromosome region 1
Small nuclear ribonucleoprotein
polypeptide N (Snrpn),
Snrpn upstream reading frame (Snurf)
Paternally expressed in the CNS 2
Paternally expressed in the CNS 3
necdin
Melanoma antigen, family L, 2
Central 7
Central 7
Maternal
Maternal
Peg12/Frat3
Central 7
Maternal
Inpp5f_v2
Distal 7
Maternal
Inpp5f_v3
Distal 7
Maternal
H19
Distal 7
Paternal
Pwcr1
Snrpn/Snurf
Pec2
Pec3
Ndn
Magel2
Mkrn3
Zfp127as/Mkrnas
Zfp127
Ring zinc-finger encoding gene 127
Ring zinc-finger encoding gene
127 antisense
Frequently rearranged in advanced
T-cell lymphomas
Inositol polyphosphate-5-phosphatase,
variant 2
Inositol polyphosphate-5-phosphatase,
variant 3
[145] (Embryonic tissues,
predicted by the embryonic lethality
of null mutations)
[142,146] (Embryonic tissues)
[147] (Embryonic tissues)
[147] (Embryonic and
extraembryonic tissues)
[148] (Early embryos and
extraembryonic tissues)
[149](Fetal brain)
[150] (Fetal brain, partially
imprinted in other fetal tissues)
[65,151] (Embryonic and
extraembryonic tissues)
[65] (Embryonic and
extraembryonic tissues)
[65,152] (Extraembryonic tissues)
[65,152] (Extraembryonic tissues)
[65,152] (Placenta-specific)
[153] (Embryonic and
extraembryonic tissues)
[154,155] (Embryonic and
extraembryonic tissues)
[150] (Embryonic tissues)
[150] (Embryonic tissues)
[156] (Embryonic and
extraembryonic tissues)
[157] (Embryonic tissues)
[158] (Embryonic tissues)
[158] (Embryonic tissues)
[134,159] (Embryonic and
extraembryonic tissues)
[160] (Embryonic tissues)
[161] (Embryonic tissues)
[161,162] (Embryonic tissues)
[163,164] (Mid-gestation
embryos, fetal brain)
[164] (Fetal brain)
[165] (Embryonic tissues)
[166–168] (Embryonic and
extraembryonic tissues)
[164] (Fetal brain)
[164] (Fetal brain)
[164,169] (Fetal brain)
[170] (Extraembryonic tissues
and fetal brain)
[171,172] (Embryonic tissues)
[173] (Pre-implantation embryo)
[174] (Embryonic tissues)
[175] (Fetal brain)
[147] (Fetal brain)
Igf2
Mpr, M6pr, Peg2, Igf-2, Igf-II
Distal 7
Maternal
Insulin-like growth factor type 2
Ins2
Mody, Ins-2, InsII, Mody4,
proinsulin, INS
Distal 7
Maternal
Insulin 2
[176,177] (Embryonic and
extraembryonic tissues)
[45] (Embryonic and
extraembryonic tissues)
[178,179] (Extraembryonic tissues)
Distal 7
Paternal
Mus musculus achaete-scute homologue 2
[135] (Placenta-specific)
Ascl2/Mash2
(continued on next page)
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F.F. Bressan et al. / Placenta 30 (2009) 823–834
Table 1 (continued )
Gene
Aliases
Chromosome
location
Preferentially
imprinted allele
Name
References
Tapa1/Cd81
Tssc4
Tspan28
Distal 7
Distal 7
Paternal
Paternal
[133] (Extraembryonic tissues)
[134,180] (Placenta-specific)
Kcnq1
Kvlqt1
Distal 7
Paternal
Cdkn1c
p57Kip2
Distal 7
Paternal
cd 81 antigen
Tumor-suppressing subchromosomal
transferable fragment 4
Potassium voltage-gated channel,
subfamily Q, member 1
Cyclin-dependent kinase inhibitor 1C
Slc22a18
Distal 7
Paternal
Solute carrier family 22, member 18
Phlda2
HET, ITM, Impt1, TSSC5, Orctl2,
Slc22a1l, Slc22a1, BWR1A
Ipl, Tssc3
Distal 7
Paternal
Nap1l4
Nap2
Distal 7
Paternal
Pleckstrin homology-like domain,
family A, member 2 (Phlda2),
Imprinted in placenta and liver (Ipl)
Nucleosome assembly protein 1-like 4
Tnfrsf23
Tnfrh1
Distal 7
Maternal
Obph1
Osbpl5
Distal 7
Paternal
Plagl1
Dcn
Lot1, Zac1
DC, PG40, PGII,
PGS2, mDcn,
DSPG2, SLRR1B
Aadc
Proximal 10
Central 10
Maternal
Paternal
Proximal 11
Maternal
Meg 1
SP2, 35 kDa, Irlgs2,
D11Ncvs75,
U2afbp-rs, Zrsr1
Meg9
Proximal 11
Proximal 11
Paternal
Maternal
Distal 12
Paternal
Dopa decarboxylase (Ddc); aromatic
L-amino acid decarboxylase (Aadc)
Growth factor receptor bound protein
U2 small nuclear ribonucleoprotein
auxiliary factor (U2AF), 35 kDa,
related sequence 1
miRNA containing gene
FA1, ZOG, pG2,
Peg9, SCP1,
Ly107, pref-1
Meg 3
Distal 12
Maternal
Delta-like 1
Distal 12
Maternal
Gene trap locus 2
Mar, Mor1, Mart1,
Peg11
Distal 12
Maternal
Retrotransposon-like 1
Distal 12
Maternal
Deiodinase, iodothyronine type III
Ddc
Grb10
U2afl1-rs1
Mirg
Dlk1
Gtl2
Rtl1
Dio3
Tumor necrosis factor receptor
superfamily, member 23
Oxysterol-binding protein 1 (Obph1),
oxysterol binding
protein-like 5 (Osbpl5)
Pleomorphic adenoma gene-like 1
Decorin
Antipeg11/Rtl1as
Hosts several miRNAs
Distal 12
Paternal
Antisense to Rtl1/Peg11
Htr2a
Htr2, Htr-2, 5-HT2A
receptor
Task3
Distal 14
Paternal
Distal 15
Paternal
Distal 15
Maternal
5-Hydroxytryptamine
(serotonin) receptor 2 A
Potassium channel, subfamily K,
member 9
Paternally expressed 13
Kcnk9
Peg13
Slc238a4
Ata3, mATA3
Distal 15
Maternal
Slc22a3
EMT, Oct3, Orct3, Slca22a3
Proximal 17
Paternal
Slc22a2
Oct2, Orct2
Proximal 17
Paternal
Igf2r
CD222, CI-MPR, Mpr300,
M6P/IGF2R
Air, Igf2ras
Proximal 17
Paternal
Proximal 17
Maternal
Airn
germline cell is protected against methylation by the zinc-finger
protein CTCF [52]. Such protection prevents interactions between
the Igf2 gene and enhancers located downstream of H19 in the
maternal allele, thus preventing Igf2 transcription. When CTCF does
not bind to the paternal allele, on the other hand, Igf2 is expressed,
and DNA is methylated within the H19 promoter region, resulting in
H19 transcriptional silencing. The different methylation status of
the Igf2–H19 locus, therefore, guarantees the exclusive paternal Igf2
expression and maternal H19 expression [51].
The importance of the parental origin of Igf2/H19 genes was
elegantly demonstrated when Kono and collaborators (2004, [54])
Solute carrier family 38,
member 4/amino
acid transport system A3
Solute carrier family 22
(organic cation transporter), member 3
Solute carrier family 22
(organic cation transporter), member 2
Insulin-like growth factor type 2 receptor
Insulin-like growth factor 2
receptor antisense RNA
[134,180,181] (Embryonic and
extraembryonic tissues)
[135,182] (Embryonic and
extraembryonic tissues)
[183,184] (Embryonic and
extraembryonic tissues)
[185,186] (Weakly in embryonic,
mainly in extraembryonic tissues)
[181] (Mainly in placenta; however,
reported not imprinted by [187])
[188] (Embryonic and
extraembryonic tissues)
[181,189] (Placenta-specific)
[151] (Embryonic tissues)
[153] (Placenta)
[190] (Embryonic heart)
[191] (Embryonic tissues)
[192] (Embryonic tissues)
[193] (Embryonic and
extraembryonic tissues)
[194,195] (Embryonic and
extraembryonic tissues)
[195,196] (Embryonic and
extraembryonic tissues)
[196] (Embryonic and
extraembryonic tissues)
[197] (Embryonic tissues and
weakly imprinted in
extraembryonic tissues)
[198] (Embryonic and
extraembryonic tissues)
[199] (Embryonic eye)
[200] (Embryonic tissues)
[157] (Embryonic and
extraembryonic tissues)
[153] (Embryonic and
extraembryonic tissues)
[201] (Placenta-specific)
[202] (Placenta-specific)
[203,204] (Embryonic and
extraembryonic tissues)
[57,205] (Embryonic and
extraembryonic tissues)
successfully produced viable parthenogenetic offspring in mice by
correcting the Igf2/H19 dosage. In this experiment, one of the
maternal alleles was derived from a non-growing oocyte (ng),
while the other was derived from a fully grown (fg) oocyte. The
process of imprinting in the maternal germline occurs at late stages
of oogenesis. Therefore, ng oocytes are considered to be ‘‘imprintneutral’’, and both H19 and Igf2 genes are expressed [43,55,56]. By
introducing a deletion in the H19 gene and its flanking regions in
the ng oocyte and consequently disrupting the imprinting of Igf2
gene, the authors demonstrated both that parthenogenetic development to term could be achieved and also that the proper
F.F. Bressan et al. / Placenta 30 (2009) 823–834
expression of Igf2/H19 likely drove modifications of other genes
that allow parthenote survival.
The Igf2r cluster, which contains Slc22a2 and Slc22a3 genes,
a solute carrier family 22 that codifies imprinted genes, is also
regulated by methylation-sensitive elements. Unlike most imprinted genes, the methylated allele is expressed in this cluster. In this
gene, the maternally methylated allele leads to paternal Igf2r
repression. The paternal non-methylated allele expresses a noncoding RNA (ncRNA), called Airn (previously named Air), which is
responsible for preventing paternal Igf2r expression [52,57].
Other important imprinted loci display the same behavior. The
Gnas and Kcnq1 loci, for example, contain ncRNAs believed to
contribute to genomic imprint control, i.e., Nespas/Gnas-as and
Kcnq1ot1, respectively. Therefore, in addition to DNA methylation
and post-translational histone modification, ncRNAs also control
imprinted gene expression [58].
The mechanisms by which ncRNAs are responsible for the
epigenetic changes observed in these imprinted loci are still not
well characterized. Numerous ncRNAs are located in clusters
regulated by ICRs [59]. In fact, each imprinted region expresses at
least one ncRNA [58,60]. Although their function and mechanisms
are not well understood, it is known that ncRNAs regulate
imprinted clusters that recruit chromatin remodeling complexes to
nearby genomic regions. The expression of specific ncRNAs, i.e.,
long ncRNAs, is associated with the acquisition of genomic
imprinting and the silencing of imprinting clusters [61,62].
827
A recently discovered imprinted retrotransposon-derived gene,
Peg10 [63], showed an essential function as an endogenous gene in
placental development [64]. Peg10 is highly conserved among
mammalian species [65], raising questions about its importance in
mammalian evolution. Ono and collaborators [64] highlighted the
possibility that ancestral mammals may have developed placenta
from newly acquired retrotransposon-derived genes or by modification of endogenous genes present in oviparous animals millions
of years ago. The understanding of the physiological roles of Peg10
and the other imprinted retrotransposon homologue Rtl1 is definitely important to improving our understanding of placental
evolution.
Disrupting the normal regulation of imprinted genes is decisive
throughout gestation and post-natal life, often leading to lethal
phenotypes in early development, as described in Table 2. Not
surprisingly, these phenotypes are related to several human
syndromes and disorders in post-natal life.
The IGF2 gene, for example, is involved in Russell–Silver
syndrome (RSS), which is characterized by the loss of methylation
in IGF2–H19 ICR, reduction in IGF2 expression, and biallelic
expression of H19, resulting in intrauterine and post-natal growth
retardation [66]. Beckwith–Wiedemann syndrome (BWS), on the
other hand, is characterized by the loss of IGF2 imprinting, causing
biallelic overexpression and a lack of expression of H19, leading to
overgrowth of the fetus, among other symptoms. Both BWS and
RSS phenotypes include pronounced growth disorders [67].
Table 2
Imprinted genes knockout and their phenotypes.
Imprinted gene
Mouse KO phenotype
References
Nesp
Gnas
Development without any obvious phenotype – behavior linked
Embryonic lethality. Heterozygous disruption is associated with significant early post-natal lethality. When maternal allele is disrupted
mice become obese. When paternal allele is disrupted, mice are hypermetabolic and thin
Increased myoclonus and deficits in motor coordination and balance
Growth retardation and early embryonic lethality due to incomplete placenta formation
Reduction in contextual fear memory, loss of hippocampal long-term potentiation
Embryonic and placental growth retardation
Neonatal lethality within 15 hours of birth, selective perturbation of late-stage differentiation structures in the epidermis
Reduction of 10–20% of weight
Embryonic and placental growth retardation, impairment of normal maternal behavior
Motor dysfunction, inducible seizures, context-dependent learning deficit
Severe post-natal growth retardation, delayed sexual maturation, but fertile. Elevated level of anxiety or fear. Motor learning deficiency,
hyperphagie
Viable offspring, with no obvious phenotypic or histopathologic defects. However, KO of its IC leads to increase in neonatal mortality and
underweight newborns showing hypotonia
Neonatal lethality and respiratory distress, underweight at birth
Reduced viability at embryonic day 12.5. Offspring showing disregulation of sleep and food intake, growth retardation soon after birth
Viable, healthy and fertile. No obvious phenotype. Triple Frat knockout (Frat1, Frat2 and Frat3) shows the same normal phenotype
Increase in placental weight, fetal overgrowth
P0 and null mutants showed reduced placental growth, followed by fetal growth restriction. Phenotypes more severe in Igf2 null
mutants at later stages of gestation
Viable and fertile, without major metabolic disorders. Ins1 and Ins2 double homozygous knockout, however, were growth-retarded,
developed diabetes mellitus and died within 48 h
Death at 10 d post-coitum, placental failure
Reduction of female fertility, increase in post-natal lethality
Deafness, circular movement and repetitive falling. Gastric hyperplasia. Severe anatomic disruption of cochlear and vestibular end
organs. Phenotypes unrelated to BWS
Divergent phenotypes in offspring. Abnormal placental development (placentomegaly and trophoblast dysplasia), morphological
defects in neonates
Placental overgrowth, consequent reduction of fetal-to-placental weight ratio
Intrauterine growth restriction, altered bone formation, increased neonatal lethality
Skin fragility, tumor development
Embryo and placenta overgrowth
Pre- and post-natal growth retardation, eyelid and skeletal abnormalities, smaller litter size, increased neonatal mortality
Fetal and post-natal growth reduction
Placental abnormalities and functional deficiencies, pre- and post-natal growth retardation, placental growth retardation, increased
late-fetal or neonatal lethality
Placental and fetal growth restriction
Impairment of neurotransmitters release. No obvious phenotypes, viable and fertile offspring
No obvious phenotypes, viable and fertile offspring
Lethality at birth, embryo overgrowth
Reduction in birth weight
[206]
[145,207]
Sgce
Peg10
Ppp1r9a
Mest/Peg1
Klf4
Kcnq1ot1
Peg3
Ube3a
Pwcr1
Snrpn
Ndn
Magel2
Peg12/Frat3
H19
Igf2
Ins2
Ascl2/Mash2
Tapa1/Cd81
Kcnq1
Cdkn1c
Phlda2
Plagl1
Dcn
Grb10
Dlk1
Gtl2
Rtl1
Slc238a4
Slc22a3
Slc22a2
Igf2r
Air
[208]
[64]
[209]
[210]
[211]
[212]
[213]
[214]
[215]
[216]
[217,218]
[219]
[220]
[221]
[26,46]
[222,223]
[224]
[225]
[226]
[227–229]
[186]
[230]
[231,232]
[233]
[234]
[235]
[196]
[236]
[202]
[237]
[238]
[57,239]
828
F.F. Bressan et al. / Placenta 30 (2009) 823–834
Abnormal imprinting patterns are also associated with neurodevelopmental disorders, such as Prader–Willi (PWS) and Angelman (AS) syndromes, which are associated with the loss of paternal
or maternal imprinting on chromosome 15q11–q13, respectively
[reviewed by [14,23,68]].
context, the generation of in vivo gene function assays is vital for
understanding the biological roles of developmental genes and
their interactions with each other and with environmental stimuli.
5. Imprinting alterations and implications
Understanding the genetic control of fetal–maternal interactions has dramatically improved with the introduction of genome
modifications in animal models. In fact, gene-targeting strategies
are the most widely accepted models used to provide reliable and
accurate information on the mechanisms of implantation and
placentation, given their ability to provide definitive evidence for
the in vivo function of a specific gene.
Genes that are candidates to have a role in early development
can have their biological effects analyzed in vivo in one of the two
ways: gain of function or loss of function studies. The first method
is based on gene overexpression, achieved by the random integration of a transgene into the genome or a targeted insertion of the
transgene into a specific locus (a knock-in). On the contrary, the loss
of function gene assay relies on the suppression of a gene function.
Mainly, it is achieved by gene-trapping in ES cells or targeted gene
deletion (a knockout, KO). The first method, although relatively
inexpensive, has the significant limitation of being only effective for
genes that are expressed in ES cells, whereas gene targeting can be
used for any gene, either permanently or in a conditional manner
[reviewed by [94–96]].
The gain of function strategy is especially interesting for characterizing placental features that are not fully described. The
transfer of transgenic embryos expressing a reporter gene, such as
green fluorescent protein (GFP) or the b-galactosidase enzyme
(LacZ), to wild-type recipients enables the precise discrimination of
uterine and trophoblast contributions to placental defects [97]. The
inverse is also valid when wild-type blastocysts are introduced into
mutant uterine tract [71]. This technique has been used for several
purposes, such as elucidating trophoblast invasion in hemochorial
placentas [98], demonstrating the spatial-temporal pattern of
imprinted gene expression in embryos [99] or revealing the X
inactivation mechanism [100,101].
KO mice model is another strategy that has greatly contributed
to the understanding of several diseases and different biological
processes [reviewed by [94]], usually revealing a gene role by
comparing the knockout phenotype with that of wild-type mice.
For example, it has been used to uncover basic mechanisms of DNA
repair [102], cancer research [103], diabetes [104], behavioral
analysis [105], and developmental related processes [106,107],
among several others.
Despite differences between mice and human morphology and
endocrine function, the mouse is the most popular model organism
for studying mammalian genomic imprinting and other processes
in eutherian animals [108]. Great advantages of mice when
compared to other animals are the availability of maternal- or
paternal-only derived embryos and the characteristics of these
animals, such as uniparental chromosomal duplications (UPD),
high fertility, low costs to maintain feeding and housing facilities,
and responsiveness to a range of assisted reproductive technologies
[94,109]. Most importantly however, is the availability of a fully
sequenced genome for this species [110] and the technology
available for the manipulation of embryonic stem cells, allowing
the use of these cells for the production of genetically altered
offspring [111,112].
The generation of KO mice relies on several in vitro procedures
that, although specific, are technically simple to perform. The first
step consists of the design and construction of the desired vector.
Circular sections of bacterial DNA (plasmids) are frequently used to
manipulate the genome of embryonic stem cells by introducing
In humans, pregnancy losses are extremely common and not
completely understood. In fact, 25% of spontaneous abortions
remain unexplained [69]. The majority of these losses occur during
the pre-implantation period, though after implantation, approximately 15–20% of pregnancies are also lost spontaneously [70,71].
In farm animals, embryonic mortality is also the major cause of
reproductive wastage, where a dysfunctional placenta accounts for
80% of this mortality [72,73].
ARTs have been widely used in an attempt to correct fertility
impairment in humans and animals and to provide a higher
reproductive efficiency in farm animals. In 2003, almost 4% of the
total number of human births in developed countries was estimated to have been produced with in vitro procedures [74]. This
scenario is not different for farm species. The last report of the IETS
(International Embryo Transfer Society), released in 2006,
announced that in the previous year, nearly 266,000 bovine
embryos were produced in vitro and transferred worldwide.
Despite its wide use, ARTs, such as IVF or cloning in animals,
increase the incidence of abnormalities in the morphology and
function of the placenta [75]. Hydroallantois, poor vascularization
and abnormal (mostly reduced but also enlarged) placentomes are
some of the most common pathological alterations [76–78].
Overall growth of the placenta and other particular structures
(such as the labyrinthine trophoblast), as well as regulation of
specific transporters and channels needed for nutrient supply to
the fetus, are frequently regulated or affected by imprinted genes
[reviewed by [25]].
Placental perturbations also lead to high birth weights and
reduced survival rates, a condition known in ruminants as large
offspring syndrome (LOS, [79,80]). This condition is reminiscent of
the BWS in humans and is correlated with IGF2R imprinting
disruption [81]. The incidence of placental failures is especially
important in cloning by nuclear transfer because such failures
represent the major cause of pregnancy failure in these animals
[76,82–85]. Placental abnormalities in cloned animals are evident
and appear frequently even in gestations carried to term [86,87].
Furthermore, the use of ARTs and their in vitro culture conditions changes the methylation and expression patterns of imprinted genes [81,88]. In laboratory animals, 5–10% of non-manipulated
embryos undergoes abnormal methylation reprogramming and
fails to develop. However, embryos derived from some kinds of
manipulation, for example, superovulation and in vitro culture,
undoubtedly present a higher rate of methylation and/or
imprinting abnormalities when compared to non-manipulated
embryos [89,90]. When nuclear transfer is considered, methylation
patterns are also abnormal and highly variable between individuals
[91,92].
Imprinted loci disruption has been observed in a number of
human developmental disorders and cancers [reviewed by [93]].
For example, a loss of imprinting (LOI) has been found in patients
with PWS (at a frequency of approximately 1%), patients with AS (at
a frequency of 3%), patients with BWS (50% of patients), and nearly
50% of the transient neonatal diabetes mellitus [reviewed by
[44,67]].
The observation that epigenetic abnormalities are present in
normal or manipulated pregnancies has made the animal model
suitable for a more profound study of these perturbations. In this
6. Transgenic strategies to study mammalian development
F.F. Bressan et al. / Placenta 30 (2009) 823–834
a DNA sequence flanked by homologous sequences into the gene to
be inactivated [113]. Reporter genes, as well as antibiotic resistance
genes, are introduced into the center of the target gene, causing
interference with expression and also allowing for the positive
selection of the transgene in the cell genome [114].
Homologous recombination of plasmid and DNA sequences is
obtained with a very low and variable efficiency rate [115]. Normally, it consists of the recombination of similar chromosome
sections derived from each parent [116]. Gene-targeting technologies exploit this characteristic by recombining transgenes containing a disrupted gene with a similar DNA sequence, leading to
targeted gene disruption.
Successfully modified embryonic stem cells are injected in preimplantation blastocoels, contributing to the tissues of the developing animal, including the germline [117,118]. Embryonic and
adult tissues are composed of transgenic and non-transgenic cells
called chimeras. Once these embryonic stem cells are integrated
into germ cells, the newly inserted gene alteration may be passed
on to the next generations. As a result, the chimeras produced are
able to generate mouse strains that are heterozygous for the altered
genes, and, most importantly, homozygous offspring can be
obtained by planned matings [reviewed by [94]].
7. Developmental studies based on knockout models
Transgenic approaches in mice have provided reliable means of
investigating complex biological phenomena or diseases by
allowing gene products to be expressed in a controlled manner in
a whole organism where the majority of the genes have a human
counterpart [119,120].
Indeed, an International Mouse Knockout Consortium
composed of four groups, the Knockout Mouse Project (KOMP,
http://knockoutmouse.or), the European Conditional Mouse
Mutagenesis Program (EUCOMM, http://www.eucomm.org), the
North American Conditional Mouse Mutagenesis Program (NorCOMM, http://norcomm.phenogenomics.ca/index.htm), and the
Texas Institute for Genomic Medicine (TIGM, http://tigm.org), was
created in 2007 to obtain a mutation of all protein-encoding genes
in the mouse using a combination of gene-targeting and genetrapping strategies [96,121].
Regarding developmental process, mouse mutants have been
created for the broad study of gene expression and developmental
interactions not only throughout the peri-implantation and gestation periods [reviewed by [1,9]] but also for different stages of
reproduction [reviewed by [107,122]].
KO models have been used for more than a decade to investigate gene function, including the role of certain genes for epigenetic patterning and embryogenesis. Trasler and collaborators in
1996 [123] showed that DNA methyltransferase (Dnmt/) KO
embryos failed to develop past the 25-somite stage and were
developmentally delayed and asynchronous. The authors
concluded that DNA methylation is vital for embryo development.
829
Five main mammalian DNA methyltransferases (Dnmt) have been
characterized and are related to the establishment and maintenance of genomic imprinting: Dnmt1, Dnmt1o, Dnmt3a, Dnmt3b
and Dnmt3L [124]. Dnmt1 and the oocyte isoform Dnmt1o are
responsible for the maintenance of the imprinted methylation
patterns [125,126], Dnmt3a and Dnmt3b are required for de novo
methylation and are essential for paternal and maternal methylation imprints during germline development [127]. Most recent
studies have shown that Dnmt3-like (Dnmt3L) cooperates with
Dnmt3a and is necessary for the establishment of genomic
imprinting during gametogenesis [128–130]. By constructing KO
mice models, it was possible to show that these methyltransferases are indispensable for embryogenesis, as summarized
in Table 3.
Similar to DNA methylation, histone modifications, mainly
acetylation and methylation, also influence gene expression
[131,132]. In contrast to embryo formation, placentation seems to
be more dependent on repressive histone methylation than DNA
methylation, as stated earlier. Some imprints in extraembryonic
tissues directly correlated with histone H3 repressive methylation
but not with DNA methylation [133,134]. In placenta, several genes
maintain imprinting status in the absence of Dnmt1 [135,136].
These genes probably have their DNA-methylated allele enriched
with histone H3-lysine-9 methylation, together with other histone
lysine methylation. Using KO models, the histone methyltransferase (HMT) G9a was shown to contribute to the allelic repression of
genes that are imprinted only in the trophoblast. The dependence
of histone post-translational modification in the parental originspecific expression probably prevents imprint erasure during the
genome-wide demethylation wave that occurs after fertilization
[29,136].
KO studies of other HMTs or histone deacetylases (HDACs) have
shown that deletions of its encoding genes (i.e., HMTs Eset and G9a
and Polycomb-group genes Ezh2 and Suz12) lead to embryonic
lethality [131,137–139]. The mechanisms by which histone modifiers regulate the maintenance of differentially allelic chromatin
organization in imprints require further investigation.
From more than 70 imprinted genes in which expression was
already reported in developmental stages, roughly half have been
analyzed through KO studies, which are summarized in Table 2. The
phenotypes observed in the KOs ranged from increased embryonic
or post-natal lethality (i.e., Gnas, Peg10, Klf4, Ascl2, Tapa1/CD81) to
no obvious phenotypes (i.e., Nesp, Peg12/Frat3, Slc22a2). Most
phenotypes evaluated by KO experiments confirm the preferential
allelic gene expression and its importance for fetoplacental growth.
For example, Table 2 shows that the deletion of the paternally
expressed genes Peg10, Mest/Peg1, Peg3, Igf2, Dlk1, Gtl2, Rtl1 and
others suppresses growth, whereas the deletion of the maternally
expressed genes H19, Grb10, Igf2r and others increases fetoplacental growth. Some mutations, although apparently unrelated to
nutrition allocation and fetal growth, are essential for fetal development, i.e., the deletion of Ube3a, Sgce, Ppp1r9a and Pwcr1, among
Table 3
DNA and histone methyltransferases knockout consequences.
Methyltransferases
Knockout consequences
References
Dnmt1
Dnmt1o
Dnmt3a
Dnmt3b
Dnmt3a and Dnmt3b
Dnmt3L
Embryonic extensive demethylation
Embryos from Dnmt1o/ females lose half of their imprints during one cell cycle
Apparently normal at birth, increased lethality at about 4 weeks of age, presenting runted phenotype
Embryonic lethality probably due to multiple developmental defects
Impaired de novo methylation. Embryonic lethality before 11.5 dpc
Null mutations reveal disruption of maternal methylation imprints. Heterozygous
progeny of homozygous females fail to develop beyond 10.5 dpc
due to abnormal development of extraembryonic structures
Decrease in H3-K9 methylation in placenta, embryonic lethality before/at 10 dpc
[240]
[126]
[127,241]
[241]
[241,242]
[128–130]
HMT G9a
[29,137]
830
F.F. Bressan et al. / Placenta 30 (2009) 823–834
others, and mainly result in impairments related to the nervous
system during the post-natal period.
Interestingly, the deletion of Peg10, a paternally expressed gene,
as well as the deletion of Ascl2, a maternally expressed gene, both
leads to embryonic lethality due to placental defects. Fetal growth
and placentation are now seen as complex processes dependent on
very particular gene expression networks. By generating animals
lacking a specific gene, it was possible to evaluate a variety of
reproductive parameters in controlled experiments, turning
transgenesis into an extremely valuable tool for imprinted gene
expression studies.
8. Conclusions and perspectives
In 2007, the Nobel Prize in physiology or medicine was awarded
Drs. Mario Capechi, Martin Evans and Oliver Smithies for their work
on genetic modifications in mice using embryonic stem cells. Great
progress in several fields of basic and medical science was made
possible with the use of animals harboring genetic modifications.
Undoubtedly, this technology has greatly contributed to the
understanding of the mechanisms that regulate genomic
imprinting and development in mammals.
For a long time, the KO approach has been the method of choice
in placentation and early development studies, allowing for the
evaluation of specific phenotypes in vivo throughout gestation.
Although this technique is well established in mice, its technical
unavailability in other animal species is a considerable drawback.
Moreover, animals other than the mice have increasingly been
accepted as research models because they may be better correlated
with human characteristics such as birth weight, organ
morphology or genome similarity (i.e., ewe, swine or primate
models). We believe that in the near future, epigenome interferences, i.e., targeted epimutations in numerous animal models, may
allow the ‘‘knockout’’ technique to become the basis for several
other new and valuable techniques in science.
By reviewing the importance of genomic imprinting in early
development in mammals and the genes involved, we have
emphasized the role of imprinted genes in successful placental and
fetal development. Moreover, we have highlighted the regulation of
some important genes, which may turn into future targets of
genetic therapies.
Because the acquisition and evolution of genomic imprinting are
among the most fundamental biological questions, further use of
gene transfer techniques to improve the understanding of this
process in mammals is warranted. In particular, gaining insight into
the regulation of epigenetic mechanisms during early development
would greatly contribute to the improvement of ARTs and their
outcomes.
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